Functional evaluation of a novel kisspeptin analogue on the reproduction of female goldfish

Kisspeptin (kp) is a key regulator of reproduction, which stimulates sexual maturation and gametogenesis in mammals, amphibians, and teleosts. In the present study, to enhance the biological activity of kp10, a novel analog (referred to as M-kp10) was designed based on the endogenous goldfish variant, in which phenylalanine 6 was substituted by tryptophan and the N-terminus was acetylated. Compared with the native kp-10 and salmon gonadotropin-releasing hormone (GnRH3), the effect of M-kp10 on sexual hormones and reproductive indices as well as the expression of kiss1, cyp19a1, and kiss1ra genes in goldfish (Carassius auratus) was investigated. In practice, peptides were synthesized based on the standard Fmoc-solid-phase peptide synthesis and purified by employing RP-HPLC, followed by approving their structure using ESI-MS. The results showed that M-kp10 increased significantly 17,20β-DHP, LH, FSH and E2 as well as fecundity, hatching and fertilization percentages than the other peptides. Histological studies revealed that M-kp10 led to the faster growth of ovarian follicles compared to the kp-10 and GnRH3. The genes of cyp19a1, kiss1ra, and kiss1 were remarkably more expressed after treatment with M-kp10. In conclusion, the results indicated the superiority of M-kp10 over kp-10 in inducing sexual maturation and accelerating the percentage of fecundity, suggesting that M-kp10 could be a promising candidate for application in the artificial breeding of fish.

Expression levels of kiss1, cyp19a1 and kiss1ra genes. All the qPCR assays described in this approach had high efficiencies (> 95% ± 0.82). The expression level of kiss1, kiss1ra, and cyp19a1 genes were assessed in the ovarian and hypothalamic tissues received M-kp10, kp-10, and GnRH3. There was a significant correlation coefficient between the relative expression of the kiss1 (p < 0.01: r = 78.44), kiss1ra (p < 0.01: r = 70. 34) and cyp19a1 genes (p < 0.01: r = 64.25) in the hypothalamic and ovarian tissues. The highest expression of these genes was recorded in hypothalamic tissue in M-kp10 treatment (p = 0.001). As indicated in Figs. 3, 4, and 5, there were significant changes in the expression levels of kiss1 (p = 0.003, F = 9.26, df = 11), kiss1ra (p = 0.002, F = 7.82, df = 11), and cyp19a1 (p = 0.002, F = 9.57, df = 11) by injecting kisspeptins and GnRH3. M-kp10 led to a much higher rise in hypothalamic kiss1 (Fig. 3), cyp19a1 (Fig. 4) and, kiss1ra (Fig. 5) mRNA levels than kp-10 compared to the control group (p = 0.001). Further, compared to M-kp10, the variations of hypothalamic kiss1, kiss1ra, and cyp19a1 mRNA levels were less significant after GnRH3 treatments. In the ovarian tissue, M-kp10 resulted in improving the expression levels of kiss1, kiss1ra, and cyp19a1 genes significantly, while fewer changes were observed following kp-10 and GnRH3 treatments.  www.nature.com/scientificreports/ Ovarian histology. Histological structure of ovary exhibited the generalized bony fish with ovary structure during 24 h post-injection ( Fig. 6A-F). Based on the microscopical examination of the female gonads of C. auratus, most oocytes were detected at the pre-vitellogenic stage (PVO) in the (A) negative control and domperidone Control (B). The oocyte number in the vitellogenic stage (VO) increased following kp-10 treatment (C), in which the yolk-filled sacs were observed between the cortical alveoli in the peripheral cytoplasm. In addition, the ovary became more developed and reached the final maturation stage (ripe oocytes) after (D) M-kp10 and (E) GnRH3 (250 μg/ml) treatments. The results reflected a greater percentage of mature oocytes in the M-kp10treated group than in the kp-10 and GnRH3-injected ones (p = 0.001, F = 28.75, df = 5) (Fig. 6G). Based on statistical analysis, mature oocytes increased in the M-kp10 group compared to the others (Fig. 6G) (p = 0.001).   (100 µg/kg) led to significant differences in the relative fecundity (p = 0.001, F = 598.81, df = 5), percentage of fertilization (p = 0.001, F = 3350.75, df = 5), and hatching (p = 0.001, F = 2154.01, df = 5) (Fig. 7). The highest fecundity was recorded in fish receiving M-kp10. Increasing in GnRH3 concentration from 100 to 250 µg/kg improved fecundity, fertilization, and hatching rates but not as much as the M-kp10 (p = 0.001).

Discussion
The members of the HPG axis are broadly used for accelerating and synchronizing oocyte maturation in the fishery industries. Given that milt amount is not considered a limiting factor in the artificial reproduction and most hormonal manipulations are utilized to increase the spawning of female fish, we designed, synthesized, and characterized a new kp-10 analog (M-kp10) to increase reproductive efficiency in female fish. Numerous kisspeptin analogs have been synthesized to improve their biological activity and/or stability, including those containing substitutions with unnatural amino acids and/or chemical modifications 13,20 . Substitution of Gly-Leu dipeptide bond, which is located at the C-terminal moiety of Kiss1 and is susceptible to proteolytic degradation with metalloproteases, improved its stability against proteolytic degradation 21 . In addition, Asami et al. reported that substitution of Arg9 improved the bioactivity compared to kp-10 and cleavage resistance 22 . The strategy of stabilizing kp-10 by N-terminal modification was indicated in kp-10 analogue C6 in  www.nature.com/scientificreports/ which an albumin binding motif inserted in the isoGlutamyl on the N-terminal amine, arginine ω-methylated at position 9, and triazole inserted between the leucine and the glycine. Moreover, C6 analogue was more active than kp-10 in ewes 23,24 . FTM080, a kisspeptin receptor agonist, indicated an increased half-life in murine serum, but lower activity than native kp-10 in ewes 25 . An interesting analogue that indicated improved serum stability was compound 26 (C26) which designed by N-terminal truncation of kp-10 26 . TAK-448 and TAK 683 are two kp-10 analogues with nine residues that exhibited comparable Kiss1 receptor-binding affinity and potency and increased half-life than kp-10 in vivo 27 . In current research, based on in vivo studies in goldfish, including analyses of sexual hormones, reproductive indices and the expression levels of kiss1, cyp19a1, and kiss1ra genes, M-kp10 promoted sexual maturation and gametogenesis more efficiently than the native kp-10. Orsini et al. reported a rise in the activity of kp-10 following substitution of Phe with Trp at C-terminus 14 . Moreover, substitution of Phe6, Arg9 and Phe10 with Ala abolished the agonistic activity of kp-10 14-16 . As a results, Phe6 and Phe9 were proposed as critical residues for binding to the hydrophobic pocket of the receptor 14 . Likewise, Gutierrez-Pascual et al. outlined the critical role of Phe6 in the agonistic activity of rat kp-10 15 . The results of current study that substitution of Phe6 with Trp improve the bioactivity of kp-10, along with previous studies underscores the important role of Phe6 in the activity of kp-10.
The limiting circulation half-life of kp-10 is an important obstacle for its application. The shorter forms of kisspeptin are less potent than the longer ones when administered peripherally due to a smaller circulation halflife 28 . Due to the limitations of production and improvement of the larger molecules, the chemical modification of the shorter molecule kp-10 is an alternative strategy to enhance the stability and activity. We speculate that the improved activity of M-kp10 can be attributable to the N-terminal acetylation, as it was shown to prevent degradation and increases their half-life in circulation 18,29 . In the present study, both strategies, i.e. amino acid replacement and chemical modification were utilized to promote the activity of kp-10 in C. auratus.
To compare the activity of kp-10, M-kp10, and synthetic GnRH3, we examined the reproductive indices, hormones level in plasma (LH, FSH, 11KT, 17-20β-DHP, E2, cortisol, and LPL), and ovarian histology as well as the expression levels of kiss1, kiss1ra, and cyp19a1 genes from the hypothalamic and ovarian tissues. The highest FSH and LH levels were observed in the M-kp10-injected group. Valipour et al. proposed that kisspeptin can play a role in secreting gonadotropins such as LH and FSH 30 . In bony fish, LH, FSH, progesterone, estradiol, and testosterone hormones stimulate oocyte growth and maturation and control these functions 31 . Whereas Li et al. reported that kp-10 cannot stimulate the secretion of LH in primary pituitary cells, current study in agreement with Somoza and colleagues suggests a direct pituitary action on LH secretion 1,10 .
E2 should be increased while enhancing FSH and 17-20β-DHP should follow while raising LH 32 . In this study increase in LH and FSH due to the injection of M-kp10 and GnRH3 (250 µg/kg) has caused an increase in E2 and 17-20β-DHP secretion.
Given that 17-20β-DHP is the main maturing hormone in fish, M-kp10 plays an important role in the final maturation of oocytes 28 . Tokumoto et al. reported that prolonged incubation with 17-20β-DHP in vivo can lead to ovulation, which reflects the role of 17-20β-DHP in oocyte maturation in freshwater fish 33 . In this study, the highest amount of 17-20β-DHP was observed in the M-kp10 and GnRH3 (250 µg/kg) groups, and also the highest percentage of oocyte maturation was observed in these two groups.
Injection of two synthetic kisspeptins into scombroid fish showed significant increases in E2 levels 34 . Significant increases in E2 levels were observed in male and female Nile tilapia exposed to the kisspeptin-10 35 . Significant increase in the levels of the E2 were observed in Chub mackerel (Scomber japonicus) that were affected by  www.nature.com/scientificreports/ kisspeptin-15 34 . In the present study, E2 levels in M-kp10 and GnRH3 (250 µg/kg) treatments showed a significant increase compared to other groups.
In the present study, the highest amount of cortisol was related to M-kp10 treatment. Further, 17-20β-DHP is involved in both the hydration and final maturation of the oocyte, while cortisol is only involved in its hydration in vitro 36 . Suzuki et al. found that a rise in cortisol during spawning catfish (loricariid catfishes) may be attributed to fish physiological activities like osmotic regulation and energy supply processes which occur at the same time as fish reproduction 37 . In this study, cortisol levels were significantly higher in the M-kp10 group compared to all other groups. Milla et al. showed that hydration of oocytes can be induced in vitro by cortisol and these data probably explain the high level of fecundity in the M-kp10 group 36 .
None of the treatments showed an increase in 11KT concentration. Plasma LPL activity increases during oocyte maturation and reaches its maximum after vitellogenesis 38 . In the present study, the treatments significantly increased the LPL activity compared to the control samples. The highest level of LPL activity in the M-kp10 group can indicate oocyte maturation and confirm the effectiveness of M-kp10.
The various effects of kisspeptin treatments on the hormones can be ascribed to the independent function of this neuropeptide in different tissues. This means that brain kisspeptin can be synthesized independently of that in ovaries and other tissues and exert its physiological role. Kisspeptin plays different physiological roles in the hypothalamic and ovarian tissues. Furthermore, the role of kisspeptins can be related to different times. The results of the previous studies have indicated that kiss1 mRNA is expressed in the fish brain and can participate in reproduction, feeding, and behavior 39 . Kiss1 has been reported in the theca and granulosa cells of the ovarian follicles in catfish 39 . According to Chang et al., kisspeptin directly affects pituitary hormone secretion in some mammals and goldfish 40 .
Expression of the kiss1 and kiss1ra genes has also been reported in brain neurons 41 as well as in organs such as the testes, ovaries, pituitary, and pancreas 42 . Kisspeptin increases the secretion of gonadotropins, but for this purpose, it must first stimulate GnRH-producing neurons in the hypothalamus, which must increase Kiss1 receptors on their cell membrane to be stimulated 43,44 . Therefore, the action of kisspeptin requires the expression and presence of kiss1ra in GnRH-producing neurons 41 . In this study, samples that were affected by M-kp10 showed a significant increase in kiss1ra gene expression in both hypothalamic and ovarian tissues compared to other groups. The results of the present study represented that M-kp10 has a higher effect on the expression of kiss1 and kiss1ra genes compared to the kp-10 and GnRH3.
Cyp19a1 gene expression has been reported in both the hypothalamic and ovarian tissues of fish 45 . Increasing the concentration of sex hormones such as E2 has increased the expression of the cyp19a1 gene in zebrafish 46,47 . The results of a study on European sea bass showed that treatment with sex hormones significantly increases the expression of the cyp19a1 gene in hypothalamic tissue 48 . Another study showed that the expression of the cyp19a1gene in the hypothalamic tissue is low until the vitellogenesis, but in the final stage of oocyte maturation, its level increases significantly 35 . In this study, the expression of the cyp19a1 gene in both hypothalamic and ovarian tissues of the group exposed to M-kp10 was significantly increased. Oocyte maturation was also high in this treatment; therefore, it seems that high levels of sex hormones and oocyte maturation in M-kp10 treatment are related to increased cyp19a1 gene expression in the hypothalamic and ovarian tissues.
One study found that injection of the 10 amino acid kisspeptin into European sea bass over 7 weeks increased gonadal growth index and sperm maturation 49 . In female clownfish (Amphiprion mel-anopus), injection of human kisspeptin increased vitellogenin synthesis and increased gonadal growth and oocyte growth over 6 weeks 50 . In chub mackerel, the concentration of kisspeptin in the final stages of oocyte maturation increased dramatically, and in Channa striatus, injection of mammalian kisspeptin increased the rate of gonadal development 51 . In the present study, groups received M-kp10 and GnRH3 (250 µg/kg) showed a significant increase in the number of oocytes in the final stage of maturation.

Conclusion
Based on the results of hormone analysis, histology, gene expression in both hypothalamic and ovarian tissues and reproductive indices, Phe6Trp substitution in parallel with N-terminal acetylation resulted in enhancing the reproductive ability of kp-10 significantly. It is suggested that our peptide could be considered as one of the alternative candidates for synthetic hormones in future studies and according to the results it seems that M-kp10 can be used to reproduce other domestic animals like sheep, goats, cattle, horses and pigs.

Materials and methods
Fish. The natural spawning season of goldfish takes place in spring and May. Therefore, the fish samples were taken in this season and the samples were in the late stages of sexual maturity. 240 female broodstock fish with an average body weight of 67 ± 5 g were supplied from a fish farm located in the North of Iran (Gilanpoor Artificial Fish Farm, Rasht, Iran). The samples were transferred to the Marine Biology Laboratory at the University of Guilan. After acclimating in a 2000-L aerated fiberglass tank for 7 days, the broodstock was randomly separated into aerated aquaria. The samples were fed twice a day with the feeding powder purchased from Isfahan Mokammel Co. (Isfahan, Iran). The number of samples in each group was 39 (three replications and each replicate: 13 fish per aquarium). The size of the aquariums was 70 × 40 × 40 cm with a volume of 112 L and the photoperiod of experiment was 14L/10D. (There were no exclusions and confounders were not controlled). In total, the number of animals that were used for hormonal and histological analysis and gene expression is shown in Table 1.

Injection and treatments.
To inject peptides into fish samples, they should be combined with a dopamine antagonist and a solvent 52 to help increase physiological efficacy, so in this study, domperidone and propylene glycol were used as a dopamine antagonist and as a solvent, respectively. The injection was performed into the muscle of the pectoral fin in one step.
To obtain the optimal dose of M-kp10, pre-test was first achieved on goldfish. The pre-test experiment was conducted with 5, 20, 50, 100, and 200 μg/kg of M-kp10 along with domperidone. The optimal dose of M-kp10 was determined as 100 μg/kg of body weight of fish. Based on Valipour et al., the optimal dose of kp-10 was 100 μg/kg of body weight 30 .

Ethics statement. This study was carried out in accordance with the recommendations in the ARRIVE
Guidelines. Based on the provided recommendation by AVMA guideline for the euthanasia and anesthesia of animals (2020), fish were anesthetized with clove oil (30 mg/l) before blood sampling. For euthanasia, the fish were first anesthetized with benzocaine and then frozen 4 . All experimental protocols were approved by the Ethics Committee in the Faculty of Science, University of Guilan (reference number 2949518).
Blood and tissue sampling. 6 h after the injection of peptides into fish 30 , following anesthesia of the fish, blood sampling was taken (5 samples per each replication) by a needle of a heparinized syringe (2 ml) was inserted into the caudal vein 53 . The blood sample was transferred into tubes and centrifuged at 3000 rpm for 10 min at 4 °C. The separated plasma was stored at −20 °C until hormonal analysis.
24 h post-injection (just before stripping the eggs by hand), the hypothalamic and ovarian tissues were separated from the dissected fish (N = 3) to measure kiss1, cyp19a1, and kiss1ra genes and histological studies of the ovarian growth 54 .
25 μL of the plasma and 50 ml of the Estradiol Biotin reagent (specific monoclonal biotinylated anti-E2 antibody) were added to the wells. After shaking for 30 s, the wells were incubated at room temperature for 30 min. In the next step, 50 μL of estradiol enzyme reagent was pipetted into each well. The mixture was shaken for the 30 s and the wells were incubated for 90 min. The washing buffer (350 μL) and 100 μL of substrate solution were then added to all wells. The wells were incubated again at room temperature for 20 min. The stop solution (50 μL) was lastly added. Finally, the wells were mixed and at the wavelength of 450 nm, the absorbance was read by the ELISA reader (ELISA reader, BioTek, ELx800, Germany). The other hormonal assays and plasma variables were measured according to the related ELISA kit protocol. 24 h after the injection, the ovulated eggs were collected by gently massaging the abdomen of fish 30 (N = 20 for each group). The eggs were weighed and counted in 1 g of eggs. Further, relative fecundity was calculated as follows 39 . www.nature.com/scientificreports/ where F illustrates relative fecundity, N indicates the number of collected eggs, and TW demonstrates the total body weight of fish (kg). For fertilization, milt was added to the eggs in a clean and dry container using the semi-dry method (100 μl of milt per 1 g of eggs). Approximately 4 h after fertilization, 100 eggs were separated from each group and the fertilization percentage was determined by a stereomicroscope (Nikon MSZ 800).

Reproductive indices.
To calculate the hatching percentage, the fertilized eggs were transferred to incubators. After four days, the hatching percentage was computed by the following formula 30 .  Histology. After fixing the ovary in Bouin's solution for 6 h and embedding with paraffin 55 , the tissue blocks were sectioned at 4-5 μm with a rotary microtome (Leica®, Wetzlar, Germany). The tissue sections were fixed on glass slides by albumin and stained with hematoxylin and eosin (H&E). An AmScope digital camera-attached Ceti England microscope was used for photographing slides 56,57 . To count and detect oocytes in each treatment, six replications were considered and 6 slides were prepared for each replicate 58 .
RNA isolation and reverse transcription for quantitative RT-PCR (qRT-PCR). Total RNA was extracted from ovarian and hypothalamic tissues with TRIzol reagent (Invitrogen, USA) according to the manufacturer's recommended protocol. The qRT-PCR was conducted for kiss1, kiss1ra, and cyp19a1 as described previously, and the actb gene was used as control. The specific primers for kiss1, kiss1ra, cyp19a1, and actb were as follows:  Statistical analysis. The data were analyzed using SPSS version 19 in Windows 10. The primary values of variables were analyzed initially assuming the normality and homogeneity of variance by Kolmogorov-Smirnov and Levene's test, respectively. The differences between various treatments were evaluated using the one-way analysis of variance (ANOVA) followed by Tukey's post hoc test to identify significant differences among the means of parameters at the confidence level of 95% and all data were expressed as mean ± SEM.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.